Immune mechanisms underlying hepatocellular carcinoma (HCC) are not well understood. Here, we show that the Toll-like receptor TLR2 inhibits production of the proinflammatory cytokine IL18 and protects mice from DEN-induced liver carcinogenesis. On this protocol, Tlr2
−/− mice exhibited more aggressive HCC development associated with impaired CD8+ T-cell function. Furthermore, Ly6ChighIL18Rα+ myeloid-derived suppressor cells (MDSC) were increased in number in the livers of Tlr2
−/− mice before tumor onset. MDSC in this setting exhibited higher iNOS levels that could inhibit IFNγ production and CD8+ T-cell proliferation in vitro. Notably, Tlr2
−/− hepatocytes produced more mature IL18 after DEN treatment that was sufficient to drive MDSC accumulation there. IL18 adminstration was sufficient to induce accumulation of MDSC, whereas hepatocyte-specific silencing of IL18 in Tlr2
−/− mice decreased the proportion of MDSC, increased the proportion of functional CD8+ T cells, and alleviated HCC progression. IL18 production was mediated by caspase-8 insofar as the decrease in its silencing was sufficient to attenuate levels of mature IL18 in Tlr2
−/− mice. Furthermore, the TLR2 agonist Pam3CSK4 inhibited both caspase-8 and IL18 expression, decreasing MDSC, increasing CD8+ T-cell function, and promoting HCC regression. Overall, our findings show how TLR2 deficiency accelerates IL18-mediated immunosuppression during liver carcinogenesis, providing new insights into immune control that may assist the design of effective immunotherapies to treat HCC. Cancer Res; 75(6); 1–10. ©2015 AACR.

Inflammation is thought to be related to tumor development, but the immune mechanisms underlying hepatocellular carcinoma (HCC) are still not well understood. In our study, we found that Toll-like receptor 2 (TLR2) inhibited production of the inflammatory cytokine interleukin-18 (IL-18) and protected mice from diethylnitrosamine (DEN)-induced HCC. Tlr2-/- mice showed a significant increase in HCC progression after DEN treatment along with impaired CD8+ T cell function. Meanwhile, hepatic Ly6Chigh myeloid-derived suppressor cells (MDSCs) were significantly increased in Tlr2-/- mice after 5 months of DEN treatment. Furthermore, MDSCs exhibited higher iNOS expression levels, which could inhibit IFN-γ production and CD8+ T cell proliferation in vitro. Hepatocytes from Tlr2-/- mice produced more mature IL-18 after DEN treatment, which caused accumulation of Ly6ChighIL-18Rα+ MDSCs in the liver. Recombinant IL-18 induced accumulation of Ly6Chigh MDSCs, and hepatocyte-specific silencing of IL-18 in Tlr2-/- mice decreased the proportion of MDSCs, increased the proportion of functional CD8+ T cells, and alleviated HCC progression. Caspase-8 was responsible for IL-18 production in Tlr2-/- mice since Tlr2-/- mice treated with caspase-8 shRNA exhibited decreased IL-18 production. Furthermore, the TLR2 agonist Pam3CSK4 inhibited both caspase-8 and IL-18 expression, decreased Ly6Chigh MDSCs, increased CD8+ T cell function, and promoted HCC regression. Our findings indicate that TLR2 deficiency accelerates IL-18-mediated immunosuppression in hepatocarcinogenesis, and TLR2 activation may alleviate HCC progression by inhibiting IL-18 production. Thus, these findings provide new insights that may be helpful in designing tumor immunotherapy.

Recently, long noncoding RNAs (lncRNA) have emerged as new gene regulators and prognostic markers in several cancers, including renal cell carcinoma (RCC). In this study, we investigated the contributions of the lncRNA MALAT1 in RCC with a specific focus on its transcriptional regulation and its interactions with Ezh2 and miR-205. We found that MALAT1 expression was higher in human RCC tissues, where it was associated with reduced patient survival. MALAT1 silencing decreased RCC cell proliferation and invasion and increased apoptosis. Mechanistic investigations showed that MALAT1 was transcriptionally activated by c-Fos and that it interacted with Ezh2. After MALAT1 silencing, E-cadherin expression was increased, whereas β-catenin expression was decreased through Ezh2. Reciprocal interaction between MALAT1 and miR-205 was also observed. Lastly, MALAT1 bound Ezh2 and oncogenesis facilitated by MALAT1 was inhibited by Ezh2 depletion, thereby blocking epithelial–mesenchymal transition via E-cadherin recovery and β-catenin downregulation. Overall, our findings illuminate how overexpression of MALAT1 confers an oncogenic function in RCC that may offer a novel theranostic marker in this disease. Cancer Res; 75(7); 1–10. ©2014 AACR.

Recently long non-coding RNAs (lncRNA) have emerged as new gene regulators and prognostic markers in several cancers including renal cell carcinoma (RCC). In this study, we investigated the contributions of the lncRNA MALAT1 in RCC with a specific focus on its transcriptional regulation and its interactions with Ezh2 and miR-205. We found that MALAT1 expression was higher in human RCC tissues where it was associated with reduced patient survival. MALAT1 silencing decreased RCC cell proliferation and invasion and increased apoptosis. Mechanistic investigations showed that MALAT1 was transcriptionally activated by c-Fos and that it interacted with Ezh2. After MALAT1 silencing, E-cadherin expression was increased while beta-catenin expression was decreased through Ezh2. Reciprocal interaction between MALAT1 and miR-205 was also observed. Lastly, MALAT1 bound Ezh2 and oncogenesis facilitated by MALAT1 was inhibited by Ezh2 depletion, thereby blocking epithelial-mesenchyme transition via E-cadherin recovery and beta-catenin downregulation. Overall, our findings illuminate how overexpression of MALAT1 confers an oncogenic function in RCC that may offer a novel theranostic marker in this disease.

ERBB3, a member of the EGFR family of receptor tyrosine kinases, has been implicated in activation of the PI3K pathway in human lung adenocarcinomas driven by EGFR mutations. We investigated the contribution of ERBB3 to the initiation, progression, and therapeutic response of EGFR-induced lung adenocarcinomas using tetracycline- and tamoxifen-inducible transgenic mouse models. Deletion of Erbb3 at the time of induction of mutant EGFR had no effect on tumorigenesis, demonstrating that ERBB3 is not required to initiate tumorigenesis. Tumors that developed in the absence of ERBB3 remained sensitive to EGFR tyrosine kinase inhibitors and retained activation of the PI3K–AKT pathway. Interestingly, acute loss of Erbb3 suppressed further growth of established EGFRL858R-mediated lung tumors. Four weeks after deletion of Erbb3, the tumors exhibited phosphorylation of EGFR, of the adaptor proteins GAB1 and GAB2, and of the downstream signaling molecules AKT and ERK, suggesting that alternative signaling pathways could compensate for loss of Erbb3. Similar to our observations with mouse tumors, we found that GAB adaptor proteins play a role in ERBB3-independent activation of the PI3K pathway by mutant EGFR in EGFR-mutant human cell lines. Finally, in such cell lines, increased levels of phosphorylation of ERBB2 or MET were associated with reduced sensitivity to acute loss of ERBB3, suggesting remarkable plasticity in the signaling pathways regulated by mutant EGFR with important therapeutic implications. Cancer Res; 75(6); 1–11. ©2015 AACR.